Programmed cell death (PCD) is integral to the development of multicellular organisms including plants. Numerous reports of plant PCD have appeared in the literature in the last 5 years and include examples that occur as part of the response to pathogen attack: e.g., the hypersensitive response (reviewed in Greenberg, et al., Proc. Natl. Acad. Sci. USA 93:12094-12097 (1996); Pennell and Lamb, et al., Plant Cell 9:1157-1168 (1997); Richberg et al., Curr. Op. Biol. 1:480-485 (1998); Lam et al., Curr. Op. Biol. 2:502-507 (1999)); the response to abiotic stress: e.g., formation of aerenchyma in hypoxic roots (reviewed in Drew, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:223-250 (1997); Drew et al., Trends Plant Sci. 5:123-127 (2000)); or as part of a normal developmental program: e.g., endosperm cell death during tracheary differentiation (Fukuda, Plant Cell 9:1147-1156 (1997); Groover and Jones, Plant Physiol. 119:375-384 (1999)), cereal seed development (Young et al., Plant Physiol. 119:737-751 (1997); Young and Gallie, Plant Mol. Biol. 39:915-926 (1999); Young and Gallie, Plant Mol. Biol. 42:397-414 (2000)), or aleurone cell death during late cereal seed germination (Kuo et al., Plant Cell 8:259-269 (1996); Bethke et al., Plant Cell 11:1033-1045 (1999); Wang et al., Plant Mol. Biol. 32:1125-1134 (1996)). During maize kernel development, the endosperm undergoes a progressive cell death that engulfs the entire tissue, leaving only the aleurone layer viable at maturity (Bartels et al., Planta 175:485-492 (1988); Kowles and Phillips, Int. Rev. Cytol. 112:97-136 (1988); Lopes and Larkins, Plant Cell 5:1383-1399 (1993); Young et al., Plant Physiol. 119:737-751 (1997); Young and Gallie, Plant Mol. Biol. 39:915-926 (1999)).
Ethylene is known to be a regulator of PCD during plant development (Campbell and Drew, Planta 157:350-357 (1983); Drew et al., Planta 147:83-88 (1979); He et al., Plant Physiol. 112:1679-1685 (1996)) and plays a role in orchestrating programmed cell death in developing cereal endosperm: exogenous ethylene can accelerate the onset of the cell death program in developing endosperm whereas inhibitors of ethylene biosynthesis or perception delay the program (Young et al., Plant Physiol. 119:737-751 (1997); Young and Gallie, Plant Mol. Biol. 39:915-926 (1999); Young and Gallie, Plant Mol. Biol. 42:397-414 (2000)). Ethylene controls many aspects of plant growth and development such as fruit development, root and leaf growth and seed germination. As shown in Figure 1, ethylene is generated from methionine by conversion of S-adenosyl-L-methionine to the cyclic amino acid 1-aminocyclopropane-1-carboxylic acid (ACC) which is facilitated by ACC synthase (Yang and Hoffman, Annu. Rev. Plant Physiol. 35:155-189 (1984)). Ethylene (C2H4) is then produced from the oxidation of ACC through the action of ACC oxidase. ACC synthase and ACC oxidase are encoded by multigene families in which individual members exhibit tissue-specific regulation and/or are induced in response to environmental and chemical stimuli. (reviewed in Fluhr and Mattoo, Crit. Rev. Plant Sci. 15: 479-523 (1996); Kende, Annu. Rev. Plant Physiol 44:283-307 (1993); Zarembinski and Theologis, Plant Mol. Biol. 26:1579-1597 (1994)).
Enzymes that degrade the compounds produced by the ethylene biosynthesis pathway are also known. Two enzymes in particular, ACC deaminase and ACC malonyl transferase, are commonly found in bacteria and can lower the concentration of ACC in the cell. ACC deaminase accomplishes this by converting ACC to α-ketobutyrate and ammonia. Nucleic acids encoding this enzyme have been used to control fruit ripening in plants (U.S. Pat. No. 5,702,933). Endogenous ACC concentration is also lowered by forming the metabolically inert compound, N-malonyl-ACC, in a reaction catalyzed by ACC N-malonyltransferase (MTase). (Liu et al., Phytochemistry 40:691-697 (1995)).
Ethylene perception involves membrane-localized receptors that, in Arabidopsis, include ETR1, ERS1, ETR2, ERS2 and EIN4 (Chang et al., Science 262:539-544 (1993); Hua et al, Science 269:1712-1714 (1995), Hua et al., Plant Cell 10:1321-1332 (1998), Sakai et al., Proc. Natl. Acad. Sci. USA 95:5812-5817 (1998)). ETR1, ETR2 and EIN4 are composed of three domains, an N-terminal ethylene binding domain (Schaller and Bleeker, Science 270:1809-1811 (1995)), a putative histidine protein kinase domain, and a C-terminal received domain whereas ERS1 and ERS2 lack the receiver domain. These genes have been grouped into two subfamilies based on homology, where ETR1 and ERS1 comprise one subfamily and ETR2, ERS2, and EIN4 comprise the other (Hua et al., Plant Cell 10:1321-1332 (1998)). These receptors exhibit sequence similarity to bacterial two-component regulators (Chang et al., Science 262:539-544 (1993)) which act as sensors and transducers of environmental signals (Parkinson and Kofoid, Annu. Rev. Genet. 26:71-112 (1992)) and as sensors in yeast and Dictyostelium that are involved in osmotic regulation (Maeda et al., Nature 369:242-245 (1994); Schuster et al., EMBO J. 15:3880-3889 (1996)).
In Arabidopsis, analysis of loss-of-function mutants has revealed that ethylene inhibits the signaling activity of these receptors and subsequently their ability to activate CTR1, a negative regulator of ethylene responses that is related to mammalian RAF-type serine/threonine kinases (Kieber et al, Cell 72:427-441 (1993)). Current understanding of the ethylene signal transduction pathway suggests that ethylene binding to the receptor inhibits its own kinase activity, resulting in decreased activity of CTR1, and consequently, an increase in EIN2 activity (which acts downstream of CTR1) that ultimately leads to an increase in ethylene responsiveness (Bleeker and Schaller, Plant Physiol. 111:653-660 (1996); Hua and Meyerowitz, Cell 72:427-441 (1998)). Differential expression of members of the ethylene receptor family has been observed, both developmentally and in response to ethylene (Hua et al., Plant Cell 10:1321-1332 (1998); Lashbrook et al., The Plant J 15:243-252 (1998)).
Because ethylene plays such a large role in plant growth and development, the identification of genes involved in the ethylene synthesis pathway is useful for creating plants with phenotypes associated with an altered ethylene-related process, such as plants having staygreen traits. The synthesis of ethylene, its perception by ethylene receptors, and its downstream signaling components have been identified in Arabidopsis and some other plant species. Prior to the advent of the present invention, however, no maize gene involved in ethylene bioysnthesis or signal transduction had been reported. Accordingly, a need exists for the identification of genes involved in the maize ethylene biosynthesis and signal transduction pathways. This invention meets this and other needs by providing, ACC oxidase, ACC deaminase, ERS1, ETR2, and EIN2 as well as methods of their use.